Wrinkles and fine lines are signs of aging. Anti-wrinkle creams make up a sizeable proportion of products intended to keep the skin youthful as long as possible. For many years, wrinkles and fine lines have been treated by moisturizing the skin, improving skin renewal, preventing degeneration of elastic fibers or promoting the synthesis of collagen.
In the category of cosmetic treatments to smooth human skin Restylane and Perlane (Q-Med, Uppsala, Sweden), which are genetically engineered hyaluronic acid derivatives, serve as a sort of collagen supplement. Collagen, the most abundant protein in the body, is an elastic polymer that makes skin firm and durable. As people age, collagen is lost from the dermal, or deeper skin layer, rendering skin more wrinkly and stiff. Retinoic acid (vitamin A) is an effective non-injection treatment for the skin, and is often used in medical treatments as well as in over-the-counter creams and lotions.
The numerous anti-aging and wrinkle creams on the cosmetic market improve the skin temporarily, but often only treat the outermost layer of skin, the epidermis. Collagen glands lie in the dermis, and so are unaffected by these creams. To circumvent this problem, alternative approaches are available. DHEA (dehydroepiandro-sterone) is a product secreted by the human adrenal glands whose serum concentration declines with aging. DHEA has indirect endocrine and intracrine actions due to conversion to androgens and estrogens. The transdermal administration of steroid hormone precursors pregnenolone, DHEA, as well as the natural hormone progesterone, has some important advantages. In recent years, a vast number of products that contain these active ingredients have been sold. An excellent composition for transporting DHEA and progesterone through the skin is an oil-in-water emulsion that contains components of the fatty tissue of the skin along with suitable permeation enhancers and use of liposomes and nanospheres.
Secretion of certain neurotransmitters, such as acetylcholine, is essential for the facial muscle contraction, and inhibition of these neurotransmitters is the basis of most of today's cosmetic formulas directed against expression wrinkles. The secretion and trafficking of proteins between compartments in eukaryotic cells is mediated by carrier vesicles that bud from one organelle and fuse with another one. This transport is mediated in part by SNARE proteins, which comprise conserved families of membrane-associated proteins such as VAMP, syntaxin and SNAP-25 families which mediate membrane fusion.
For example, in the synaptic transmission in response to Ca2+ influx, synaptic vesicles containing neurotransmitters fuse with the presynaptic membrane and release their contents into the synaptic cleft. Similarly, at the neuromuscular junction, Ca2+ stimulates the exocytosis of presynaptic vesicles containing acetylcholine, which is released into the synaptic cleft. SNARE proteins such as VAMP, syntaxin and SNAP25 are involved in the exocytosis of these vesicles, which contain different neurotransmitters, such as catecholamines (norepinephrine/noradrenaline, epinephrine, and dopamine), acetylcholine, serotonin and/or peptide neurotransmitters, such as enkephalins.
Acetylcholine is one very important neurotransmitter. Release of acetylcholine is important for muscle contraction. Acetylcholine may also be involved in nocioception and chemosensitivity. The inhibition of acetylcholine release may be one method for preventing those muscle contractions that lead to increased facial expression wrinkles.
Most of today's cosmetic formulas directed against facial expression wrinkles are based on the inhibition of acetylcholine secretion which is essential for facial muscle contraction. Botulinum toxin is among the most commonly used molecules against expression wrinkles, mainly for the wrinkles between the eyebrows, at the periphery of the eyes and on the forehead. Even though most botulinum toxins were found to be safe for use in humans, administration of these agents by intra-muscular injections remains an inconvenient method of administration, is immunogenic and can have some adverse effects.
The most frequently reported adverse events in clinical trials for botulinum toxin A (BOTOX®) were dysphagia (19%), upper respiratory infection (12%), neck pain (11%), and headache (11%). Caution should be exercised when administering BOTOX® to individuals with periphery motor neuropathic disease (e.g., amyotrophic lateral sclerosis, motor neuropathy) or neuromuscular junctional disorders (e.g., myasthenia gravis). Furthermore, it cannot be excluded that such toxins could be stored in the central nervous system for long period of time. In any case, botulinum toxin is highly immunogenic.
More recently, to circumvent the intra-muscular injection of toxins, transdermal botulinum toxin compositions and administrations have been described (U.S. Patent Publication 20050074461; U.S. Patent Publication 20050175636). However, presence of a toxin on the skin could be hazardous, due to possible contact with the mouth, nasopharyngeal region and the digestive tract.
While numerous studies have addressed the mode of action of botulinum toxin, many of the aspects have still to be uncovered. BOTOX®, for example, comprises three distinct biological regions, one mediating cell-specific binding, a second mediating cell entry, and a third having protease activity towards various substrates (e.g., SNARE complex proteins VAMP, syntaxin and SNAP-25) (Montecucco et al., Trends Biochem. 18:324-327, 1993). Botulinum toxins specifically cleave proteins present in the SNARE complex. These SNARE proteins are involved in the exocytosis of vesicles containing acetylcholine. In the presence of BOTOX®, the last nine amino acids of SNAP25 are removed. This truncated form of SNAP25 is extremely stable and competes with wild type SNAP25. Similarly, VAMP or syntaxin can be cleaved by other classes of botulinum toxins.
An attractive alternative to Botox comprises the production of truncated forms of SNAP25, VAMP and syntaxin which exhibit activities similar to those of botulinum toxin, or to peptides designed to specifically block the release of acetylcholine by acting on the vesicles involved in the exocytosis (the SNARE complex). A peptide (Acetyl hexapeptide-3) (Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2) (SEQ ID NO: 24) that mimics the C-terminal sequence of SNAP-25 has been shown to inhibit secretory vesicle docking in chromaffin cells (Gutierrez et al., J. Biol. Chem. 272:2634-2639, 1997). This peptide also affects the release of neurotransmitters by blocking exocytosis. This acetyl hexapeptide (also named ARGIRELINE®, Lipotec SA, Barcelona, Spain) is an anti-wrinkle peptide, which acts through a unique mechanism that relaxes facial tension and leads to a reduction in superficial facial lines and wrinkles. A study published in the International Journal of Cosmetic Science used a 10% concentration of the peptide in an oil/water emulsion. The results showed reduced wrinkle depth up to 30% after a 30-day treatment (V. Kanga, Skin care delivery systems, Happi, 47 (January 2004); International Journal of Cosmetic Science 24:303, 2002). Thus far, the SNAP25 molecule was the target for blocking neurotransmitter release either for BOTOX® or for ARGIRELINE®. The former agent is used in injection, the latter is used as topical.
Based on peptide sequences, the palmitoylated pentapeptide (Pal-KKTTS) (SEQ ID NO: 25) could have a beneficial effect on the skin. Palmitoyl oligo-peptide is a lipophilic modified peptide developed by Sederma SA and owned by Croda and is marketed as MATRIXYL®. Pentapeptides are composed of the amino acids lysine, threonine and serine, which are made lipophilic by the attachment of palmitic acid. This leads to the sequence Pal-Lys-Thr-Thr-Lys-Ser or Pal-KTTKS (SEQ ID NO: 26), a combination that mimics nature's tissue-regenerating processes by signaling the cells of the dermis to synthesize proteins (collagen I, III, IV) and polysaccharides (glycoaminoglycanes, hyaluronic acid) which make up the connective tissue necessary for padding the skin (U.S. Pat. No. 6,620,419). Two teams of investigators say palmitoyl pentapeptide is as effective as retinol in improving the effects of photo-aging but without the side effects often associated with retinol. These were two separate trials supported by Sederma SA. The first study examined the effects of palmitoyl pentapeptide (3 ppm) versus retinol (700 ppm) to the crow's feet area on photo-aged skin. The second study examined the effects of palmitoyl pentapeptide (5 ppm) on the structure of elastin and collagen IV in women with photo-aged skin. Palmitoyl pentapeptide triggered growth in elastin and collagen IV and enhanced the structure of elastin and collagen IV. The peptide was not associated with any side effects and was a safe and potent alternative to retinoids in wrinkle repair.
Oligopeptides obtained by the biotransformation of native proteins from the seeds of Hibiscus esculents L. (okra) is a patented complex in Myoxinol LS 9736 (Cognis, Cincinnati, Ohio). It is primarily composed of low molecular weight oligopeptides, allowing good bioavailability. These botanical peptides combat wrinkles in a similar way to botulinum toxin, by inhibiting the mechanical factors responsible for the appearance of expression lines on the face. This novel active ingredient has a dual action, working biologically to retard the aging of cells (anti-free radical activity), and mechanically to inhibit facial muscle contraction. Botulinum toxin prevents the formation of dynamic wrinkles such as horizontal and vertical frown lines across the forehead, crow's feet around the eyes and naso-labial lines around the mouth. The ingredient's potential as an anti-wrinkle agent was measured using an in vitro test on contraction of innervated muscle cells. The ingredient's ability to inhibit the spontaneous contraction of muscle cells was evaluated by recording the frequency of contractions over 24 hours using Carisoprodol, a known muscle relaxant, as a positive control. Cognis reported that a cream containing 1% Myoxinol LS 9736 applied to the crow's feet area over a three-week period suggested considerable anti-aging properties. The cream resulted in smoother skin, and wrinkles were 26% less noticeable due to significant reduction in muscle cell contractions, the primary mechanical factor responsible for the appearance of dynamic facial wrinkles. The contraction-inhibiting effect stops 24 hours after application.
Other synthetic peptides that mimic the amino acid sequence of segments from SNARE proteins have been used to investigate the functional role of proteins implicated in the secretory pathway, such as ESUP-A (Gutierrez et al., J. Biol. Chem. 272:2634-2639, 1997). Often the use of the synthetic peptide is limited by the difficulty in entering the target cell and an artificial system requiring permeabilization of the cell by saponin is necessary to mediate an effect of the synthetic peptide. This problem could be circumvented by fusing synthetic peptides to a domain known to translocate peptide or protein into cells such as known protein translocation domains (PTD). PTDs, such as a segment of TAT protein of HIV have been extensively studied and are known as efficient approach to transduce proteins into different cell type (for a review see Dietz et al., Mol. Cell. Neurosci. 27: 85-131, 2004). The basic transduction domain of HIV has also been shown to mediate translocation into various organs of mice. Intraperitoneal or intravenous injection of the PTD fusion protein resulted in delivery of the protein in various organs, including brain. On the contrary, there is only one example of the topical penetration of a catalase protein when fused to TAT PTD or to 9-Arginine (9R) (Jin et al., Free Radical Biology and Medicine, 31:1509-1519, 2001). However, no effect on the release of neurotransmitter was shown when the TAT PTD or 9-Arginine (9R) was used alone. Similarly, 9-polylysine enhanced penetration of superoxide dismutase into mammalian skin (Park et al., Molecules and Cells 13:202-208, 2002).
Aside from the SNAP 25 sequence, no other peptides have been reported to block neurotransmitter release. U.S. Pat. No. 6,794,362 describes use of peptides derived from the elastin protein for use in cosmetic compositions. U.S. Pat. No. 6,358,929 describes use of Lysine-Proline-Valine (KPV) peptides and derivatives as additives in a cosmetic composition intended to suppress or reduce contact hypersensitivity reactions. U.S. Pat. No. 6,183,759 describes synthesis of peptides conjugated to lanolin-derived non-hydroxyl fatty acids for use in cosmetic compositions.
Additionally, the binding of acetylcholine to its receptor on the muscle cell triggers muscle contraction. The contraction of a muscle involves transient interaction of myosin and actin. Myosin is organized in thick filaments whereas actin is polymerized in thin filaments (F-actin). Muscle contraction occurs by the sliding of the thin and the thick filaments past each other. The assembly also includes minor muscle proteins a-actinin, desmin, vimentin and nebulin. Alteration in the filaments formation, such as alteration of the actin polymerization could be one way to avoid muscle contraction and provoke muscle relaxation.
In injured muscles, wound healing is often retarded by a fibrotic process. Specific growth factors such as TGF-β are involved in this fibrotic process. Overexpression of TGF-β leads to events which cause increased deposit of matrix proteins leading to fibrosis. Activation/processing of TGF-β by specific enzymes such as the proconvertases (e.g. furin among others) is a key event in the fibrotic process. Blocking the activation of TGF-β via proconvertases could be beneficial in wound healing.
Thus, there remains a need in the art to identify peptide sequences that have pleiotropic functions such as inhibiting neurotransmitter release and inhibiting muscle contraction, and simultaneously provoking muscle relaxation, and are useful as a treatment for wrinkles and for injured muscles.